Under this grant we have discovered a number of G protein binding proteins, established their properties and revealed new roles for heterotrimeric G proteins in cells. During the current funding period we characterized GIV, a non-receptor GEF that activates G?I proteins. GIV serves as a multimodular signal transducer that assembles protein complexes and links G? subunits to other signaling pathways (growth factor and PI3K/Akt signaling) and to several important cell functions, including cell migration, autophagy, transport of secretory proteins, and endocytosis of growth factor receptors. GIV also binds G?s, promotes EGF receptor downregulation, and shuts down proliferative signaling from endosomes. The overall goal of the work proposed is to take advantage of the tools and insights we have developed to shed light on some well-known but poorly understood functions of G proteins especially on intracellular membranes. We will focus on two specific aims: Specific Aim #1: To pinpoint the mechanisms by which GIV and its interactions with G?I and G?s regulate EGFR trafficking and growth factor signaling. Our working model based on preliminary data is that GIV sequentially binds G?i3 and G?s after EGF stimulation and that phosphomodification of GIV regulates the relative binding to the two G proteins and determines whether cells migrate or proliferate. Specific Aim #2: To determine the role of G?i3 and GIV in Golgi functions and to define the mechanisms involved. Our data indicate that depletion of GIV or a GIV mutant that fails to activate G?i3 slows transport of newly synthesized proteins through the Golgi. Our working hypothesis based on preliminary data is that GIV does so by activating G?i3, down-regulating Arf1 activity, and facilitating vesicle uncoating. These studies can be expected to provide novel insights into how GIV binds and regulates two different G proteins and influences growth factor signaling, Golgi functions, cell migration and cell proliferation. Based on its crucil functions in regulating cell behavior, it is essential to define GIV's functions and interactions. Our studies will not only provide insights into fundamental roles of G proteins in cell functions, but also it may establish new paradigms that will help to identify new therapeutic targets for development of pharmacologic and anti-tumor agents. We are uniquely positioned to tackle these problems by taking advantage of the new tools, expertise, and information we have in hand.